2.5.1.1 The indirect avidin-biotin complex (ABC) method
Immunostaining of longitudinal nerve sections, dorsal root ganglia and skin was carried
out according to the indirect avidin-biotin complex (ABC) peroxidase nickel
enhancement procedure (Shu et al., 1988). Immunoreactive structures show as black permanent staining against a well contrasted pale background, which is optimal for
image analysis (Terenghi and Polak, 1994). A known positive control was included in
each batch o f staining to ensure reliability of the technique (Emson, 1993).
Sections were cut on the cryostat, collected on slides coated with Vectabond (Vector Laboratories, Peterborough, UK) and air-dried for 4h at room temperature. The
thickness of the sections was decided according to the type o f tissue. Longitudinal nerve
sections were cut at 15pm, skin at 20pm and DRG at 20pm. The slides were then
wrapped in aluminium foil and stored at -20°C overnight. On the day of staining, the
sections were thawed and allowed to air-dry for a minimum period of 4 hours. The
contours of the sections were outlined with DAKO pen (DAKO A/S, Denmark) to limit
the antibody solutions to the sections and prevent flooding o f the slide. The sections
were initially immersed in 0.2% phosphate-buffered Triton X for one hour. Any
endogenous peroxidase was then inhibited by immersing the sections in 0.05%
hydrogen peroxide (30% solution, BDH Laboratories, UK) in PBS for 20 min at room
temperature. The sections were washed three times in plain PBS followed by incubation
for 15 min at room temperature with normal serum (dilution 1:30 in PBS). This serum
was derived from the same species as the second layer antibody and was used to block
antibodies (ICN, UK). Following incubation, the normal serum was drained and the
appropriate first layer antibody was applied as approximately lOOpl per section (cf.
2.5.1.2). The sections were incubated overnight at 4°C, following which they were first
washed by immersion in Tween/PBS (dilution 1:4000, Polyoxyethylenesorbitan
monolaurate. Sigma, USA) for one min to remove unbound primary antibody, then
three times in plain PBS. The second layer biotinylated antibody (cf. 2.5.1.2) was
applied and incubated for an hour at room temperature in a humid chamber. A further
rinse in Tween/PBS for one min was followed by three more washes in plain PBS.
Peroxidase conjugated avidin-biotin (ABC, Vectabond, UK) was applied to the sections
and incubated for an hour at room temperature in a humid chamber. The sections were
finally washed in PBS before being equilibrated in O.IM sodium acetate buffer for 10
min. The slides were then immersed in a developing solution containing 7.25g
ammonium nickel sulphate (Sigma, USA), 125mg 3,3 diaminobenzidine tetrachloride
dihydrate (Sigma, USA), and 600mg a (+) D glucose (Sigma, USA) dissolved in 300ml
of O.IM sodium acetate buffer pH 6.0. The solution was activated by the addition of 6mg glucose oxidase (Sigma, USA) immediately prior to immersion of the slides for
stain development. The time required for development was determined by the degree of
staining which was monitored by examination under a light microscope of the known
control slide included in each batch, and as a norm spanned between 5 and 10 min
dependant on primary antibody used. After adequate development of immunostaining,
the reaction was arrested by immersion in O.IM sodium acetate buffer pH 6.0. The
sections were then dehydrated through graded alcohols (industrial methylated spirit,
BDH Laboratories, UK), cleared in xylene (BDH Laboratories, UK) before being
2.5.1.2 Antibodies
A panel of antibody markers were used in this study to assess the volume and rate of
axonal regeneration, the proliferation density o f Schwann cells, the macrophage
infiltration and the pattern of angiogenesis within the nerve grafts and conduits.
Antibodies to neural peptides and proteins were also used to assess nerve terminal
density in skin and neuronal cell bodies in the dorsal root ganglia. The antibodies
reacted with known, specific antigens as described below and were applied at exact
dilutions as summarised in Table 2.1 :
Table 2.1
First layer antibodies and their specific dilutions used in indirect ABC
immunohistochemistry.
Antibody Type Source Dilution
PGP 9.5 Rabbit polyclonal Affiniti 1:5000 CGRP Rabbit polyclonal Affiniti 1:8000 S-100 Rabbit polyclonal Dako A/S 1:8000 ED-1 Mouse monoclonal Serotec 1:600 VWf Rabbit polyclonal Dako A/S 1:5000
Protein gene product 9.5 (PGP) is a neuron-specific cytoplasmic protein (Jackson and Thompson, 1981). Antisera to this marker demonstrates all types of nerve fibres
pan-neuronal marker for the demonstration of cutaneous nerves (Karanth et al., 1991; Kennedy and Wendelschafer-Crabb, 1993) allowing an accurate assessment of
cutaneous reinnervation (Dalsgaard et al., 1989).
Calcitonin gene-related peptide (CGRP) is a 37 amino-acid peptide formed by tissue specific alternative splicing of the calcitonin gene product (Morris et al., 1984). It is found in small myelinated and unmyelinated AÔ and C sensory fibres (Kruger et al., 1989) and is generally the first neuropeptide to be detected in regenerating nerves (Zbao
et a l, 1993; Iwasbita et al., 1994). It is anterogradely transported and accumulates in the distal ends of regenerating axons and is bence clearly visible in the regenerating front of
injured nerves (Ekstrom and Tomlinson, 1989), as well as in neuronal cell bodies.
S-100 is a specific Schwann cell cytoplasmic antigen (Moore, 1965; Donato, 1986). The antiserum was used to identify the presence of Schwann cells within the graft or
conduit, their progression and proliferation density.
ED-1 is a monoclonal marker specific for macrophages. The ED-1 antibody recognises a single chain glycoprotein expressed predominantly on the lysosomal membrane of
tissue macrophages (Dijkstra et al., 1985; Damoiseaux et al., 1994). It was used to estimate the infiltration of the graft or conduit by macrophages, as an indication of the
inflammatory response.
Von Willebrand Factor (vWfi, previously known as factor Vlll-related antigen is isolated from human plasma. The antibody reacts with vW f and positive staining is
demonstrated in endothelial cells and megakaryocytes (Sebested and Hou-Jensen, 1981;
demonstrated the pattern of angiogenesis and infiltration of the conduits by new
capillaries.
The second layer biotinylated antibodies used were dependant on the species of origin
of the first layer antibody, whether polyclonal or monoclonal, and are summarised in the
following table (Table 2.2)
Table 2.2
Second layer antibodies and
immunohistochemistry.
their specific dilutions used in indirect ABC
Antibody Type Source Dilution
Biotin conjugated anti-rabbit Goat polyclonal Vector 1:100
Biotin conjugated anti-mouse Horse monoclonal Vector 1:100
2.5.2 Thionin blue and acridine orange staining
Semithin (1pm) transverse sections of the distal superficial radial nerve in the cat and
the distal sciatic nerve in the rat were cut on a Reichert-Jung Ultracut E ultramicrotome,
floated onto distilled water, collected on poly-L-lysine coated glass slides and dried on
a hot plate. The sections were stained with thionin blue and counterstained with
acridine orange to enhance the contrast of the myelin sheaths. Complete section
few seconds prior to staining. The dried sections were stained with freshly-made filtered
thionin^ (Thionine, Sigma, USA; 0.05g thionin in 22,5 ml absolute alcohol and 5ml
O.IM sodium hydroxide) for 45-60 seconds at 70°C on a hot plate. The slides were then
washed in distilled water and stained on the hot plate at 70°C for 30 seconds with
acridine orange (Acridine orange, BDH Laboratories, UK; 0.25g acridine orange added
to 20ml distilled water and 5ml O.IM sodium hydroxide). Following a further wash in
distilled water, the sections were finally dried on a hot plate and mounted in DPX.